Abstract
Multiphoton microscopy (MPM) can non-invasively measure the dynamic biochemical properties deep in scattering biological samples and has the potential to accelerate clinical research with advances in deep tissue imaging. However, in most samples, the imaging depth of MPM is limited to fractions of a millimeter due to blurring caused by refractive index mismatching throughout tissue and background fluorescence, overshadowing the signal in conventional MPM. To overcome these challenges, we developed a novel 3D adaptive optics (AO) system that uses an interpolated network of endogenous guide stars to focus laser light more efficiently into highly scattering samples. The synergistic combination of our AO system with DIVER detection technology enables millimeter-scale imaging with diffraction-limited resolution with optimization times between 15 s and 65 s. We characterized the algorithm and wavefront interpolation performance in a flat 2D sample and in 3D using fluorescent beads embedded in gels of various optical heterogeneity. We also tested the system in biological tissue, improving image brightness by a factor of 5 at depths of ∼0.4 mm in the fresh green fluorescent protein-tagged mouse skin and ∼2 mm in a formalin-fixed yellow fluorescent protein-tagged mouse brain. By collecting forward and back-scattered fluorescence light to optimize the excitation wavefront, AO DIVER allows imaging of the tissue architecture at depths that are inaccessible to conventional multiphoton microscopes.
Highlights
Deep tissue imaging has the potential to transform biology and medicine by visualizing the interactions between cells and their environments on a tissue-wide scale
To expand the technique to enable three-dimensional imaging on millimeter scales with a single deformable mirror, we developed a user interface to generate a network of endogenous guide stars
To improve the focusing of excitation light using a high-speed (∼40 kHz) segmented MEMS deformable mirror (Boston Micromachines Hex-111, Cambridge, MA), we implemented a feedback loop (Fig. 3) that uses the intensity of a guide star as a measure of the validity of a wavefront correction
Summary
Deep tissue imaging has the potential to transform biology and medicine by visualizing the interactions between cells and their environments on a tissue-wide scale. One of the most promising imaging modalities for non-invasive deep tissue imaging with submicrometer resolution is multiphoton microscopy (MPM).. By using a pulsed infrared laser to image deep into samples, MPM causes less phototoxicity and suffers less from image blurring compared to the shorter wavelengths of light used in confocal microscopy.. Scattering and absorption of the infrared excitation light used in MPM are significantly smaller in comparison with the shorter visible wavelengths used in confocal microscopy.. Thicker sections of tissue can be imaged due to the improved depth penetration of longer wavelengths, allowing for more accurate measurements of morphology and physical properties.. Even in highly scattering samples, the spatio-temporal focusing requirements of nonlinear signal generation mitigate the blurring effect of scattering. Scattering and absorption of the infrared excitation light used in MPM are significantly smaller in comparison with the shorter visible wavelengths used in confocal microscopy. Thicker sections of tissue can be imaged due to the improved depth penetration of longer wavelengths, allowing for more accurate measurements of morphology and physical properties. Even in highly scattering samples, the spatio-temporal focusing requirements of nonlinear signal generation mitigate the blurring effect of scattering.
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